Members of the IFN regulatory factor (IRF) family regulate gene expression critical to immune response, hemopoiesis, and proliferation. Although related by homology at their N-terminal DNA-binding domain, they display individual functional properties. The distinct properties result from differences in regulated expression, response to activating signals, and interaction with DNA regulatory elements. IRF-3 is expressed ubiquitously and is activated by serine phosphorylation in response to viral infection or TLR signaling. Evidence indicates that the kinases TANK-binding kinase 1 and inhibitor of NF-κB kinase-ε specifically phosphorylate and thereby activate IRF-3. We evaluated the contribution of another member of the IRF family, IRF-5, during viral infection since prior studies provided varied results. Analysis of phosphorylation, nuclear translocation, dimerization, binding to CREB-binding protein, recognition of DNA, and induction of gene expression were used comparatively with IRF-3 as a measure of IRF-5 activation. IRF-5 was not activated by viral infection; however, expression of TANK-binding kinase 1 or inhibitor of NF-κB kinase-ε did provide clear activation of IRF-5. IRF-5 is therefore distinct in its activation profile from IRF-3. However, similar to the biological effects of IRF-3 activation, a constitutively active mutation of IRF-5 promoted apoptosis. The apoptosis was inhibited by expression of Bcl-xL but not a dominant-negative mutation of the Fas-associated death domain. These studies support the distinct activation profiles of IRF-3 in comparison to IRF-5, but reveal a potential shared biological effect.

The thyroid hormone receptor (TR) recruits the nuclear corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), to target DNA elements in the absence of ligand. While the TR preferentially recruits NCoR, the mechanism remains unclear. The corepressors interact with the TR via interacting domains (IDs) present in their C terminus which contain a conserved motif termed a CoRNR box. Despite their similarity, the corepressor IDs allow for nuclear receptor specificity. Here we demonstrate that NCoR stabilizes the TR homodimer when bound to DNA by preventing its dissociation from thyroid hormone response elements. This suggests that NCoR acts to hold the repression complex in place on target elements. The TR homodimer recruits NCoR through two of its three IDs, one of which is not present in SMRT. This unique ID, N3, contains a CoRNR box but lacks the extended helical motif present in each of the other IDs. Instead, N3 contains an isoleucine just proximal to this motif. This isoleucine is also conserved in N2 but not in the corresponding S2 domain in SMRT. On thyroid hormone response elements and in mammalian cells this residue is critical in both N3 and N2 for high-affinity TR binding. In addition, this residue also controls specificity for the interactions of TR with NCoR. Together these data suggest that the specific recruitment of NCoR by the TR through a unique motif allows for stabilization of the repression complex on target elements.

The thyroid hormone receptor (TR) and retinoic acid receptor (RAR) isoforms interact with the nuclear corepressors [NCoR (nuclear corepressor protein) and SMRT (silencing mediator for retinoid and thyroid hormone receptors)] in the absence of ligand to silence transcription. NCoR and SMRT contain C-terminal nuclear hormone receptor (NHR) interacting domains that each contain variations of the consensus sequence I/L-x-x-I/V-I (CoRNR box). We have previously demonstrated that TRbeta1 preferentially interacts with NCoR, whereas RARalpha prefers SMRT. Here, we demonstrate that this is due, in part, to the presence of a novel NCoR interacting domain, termed N3, upstream of the previously described domains. An analogous domain is not present in SMRT. This domain is specific for TR and interacts poorly with RAR. Our data suggest that the presence of two corepressor interacting domains are necessary for full interactions with nuclear receptors in cells. Interestingly, mutation of N3 alone specifically decreases binding of NCoR to TR in cells but does not decrease NCoR-RAR interactions. In addition, while the exact CoRNR box sequence of a SMRT interacting domain is critical for recruitment of SMRT by RAR, the CoRNR box sequences themselves do not explain the strong interaction of the N2 domain with TRbeta1. Additional regions distal to the CoRNR box sequence are needed for optimal binding. Thus, through sequence differences in known interacting domains and the presence of a newly identified interacting domain, NCoR is able to preferentially bind TRbeta1. These preferences are likely to be important in corepressor action in vivo.

Nuclear receptor corepressor (NCoR) mediates transcriptional repression by unliganded nuclear receptors and certain steroid hormone receptors (SHRs) bound to nonphysiological antagonists, but has not been found to regulate SHRs bound to their natural ligands. This report demonstrates that NCoR interacts directly with the androgen receptor (AR) and represses dihydrotestosterone-stimulated AR transcriptional activity. The NCoR C terminus, containing the receptor interacting domains, was necessary for repression, which was ablated by mutations in the corepressor nuclear receptor (CoRNR) boxes. In contrast, the NCoR N terminus, containing domains that can recruit histone deacetylases, was not necessary for repression. Binding studies in vitro with a series of glutathione-S-transferase-NCoR and -AR fusion proteins demonstrated a direct interaction that was similarly dependent upon the NCoR corepressor nuclear receptor boxes and AR ligand binding domain and was independent of ligand and helix 12 in the AR ligand binding domain. This NCoR-AR interaction was further demonstrated in mammalian two-hybrid assays and by coimmunoprecipitation of the endogenous proteins from a prostate cancer cell line. Finally, AR transcriptional activity could be enhanced in vivo by sequestration of endogenous NCoR with unliganded thyroid hormone receptor. These results demonstrate that AR, in contrast to other SHRs, is regulated by NCoR and suggest the possibility of developing selective AR modulators that enhance this interaction.

Our results suggest that Her2 and Ki67 immunohistochemistry and the presence of intraductal necrosis aid in DCIS risk stratification.

S S S t t t o o o n n n y y y B B B r r r o o o o o o k k k U U U n n n i i i v v v e e e r r r s s s i i i t t t y y yThe official electronic file of this thesis or dissertation is maintained by the University Libraries on behalf of The Graduate School at Stony Brook University. These studies indicate a significant link between two host survival mechanisms -the DNA damage response and the response to viral infection. © © © A A A l l l l l l R R R i i i g g g h h h t t t s s s R